The factor for release of the human growth hormone, also called somatocrinine (hereinafter designated by the abbreviation "hGRF") is a positive regulator for the secretion of growth hormone (hereinafter designated by the abbreviation "GH") by the adenohypophysis. hGRF has been isolated from a human pancreatic tumor causing acromegaly; it is a polypeptide of 44 amino acids which has been sequenced (Guillemin et al., Science 218, 585-587, 1982). Antibodies against this peptide have enabled identification of an immunoreactive material in the hypothalamus of various primates and an apparently identical polypeptide has also been isolated from the human hypothalamus. Finally, experiments of the "southern blot" type indicate that there is only a single gene coding for hGRF. This, therefore, suggests that the tumoral pancreatic factor is coded by the same mRNA as the physiological hypothalamic factor. The usefulness of being able to produce substantial amounts of human GRF is due in particular to the fact that its genetic or physiological deficiency is a cause of dwarfism; that its stimulating effect on the synthesis of growth hormone (GH) should lead to its usefulness in diagnosis of deficiencies or disorders of GH metabolism; that its administration could provide an acceleration in regeneration of tissue, for example, in the treatment of severe burn victims and that it has been shown that the administration of human GRF to animals stimulates their growth.
A form of hGRF having only 40 amino acids residues has also been reported and fragments of GRF having as few as about 27 amino acid residues have been found to be biologically active. The full 44 amino acid residue pancreatic hGRF peptide has been synthesized chemically, and synthetic hGRF, synthetic hGRF fragments, and synthetic analogs thereof represent a potential source of highly potent regulatory substances; however, chemical synthesis of such long peptide chains is quite expensive.
Recombinant DNA techniques by which hGRF DNA is introduced into a cell to express hGRF have also been developed .
However these techniques yield only rather limited amounts of hGRF.
Belgian Pat. No. 898.666 describes such a technique wherein chemically synthesized DNA fragments are combined to DNA fragments isolated from natural source.
The process of the present invention is inspired from the method consisting in producing fused or hybrid proteins from which the desired protein itself is then eventually recovered.
Examples of such methods are
the European Patent Application Publication No. 0 020 290 and the German Patent application No. 2922496 which describe the preparation of a protein by selective enzymatic cleavage of the C-terminal from a fusion protein with the N-terminal of a particular tetrapeptide sequence;
the European Patent Application Publication No. 0 133 282 which describes the preparation of a polypeptide amide such as growth hormone releasing factor by producing a glycine peptide by genetic technology and enzymatically removing the C-terminal glycine;
the Australian Patent Application No. 85/37071 which describes the preparation of a fibrinolytically active hybrid protein by different methods among which the method comprising taking the genetic information of each protein, cutting and ligating this to construct a DNA sequence coding for the hybrid protein and expressing this DNA in prokaryote or eukaryote hosts, the separate chains being eventually prepared thereafter by mild reduction to break interchain disulfide bridges followed by affinity chromatography;
the European Patent Application Publication No. 0 134 085 which describes the production of prepro-human pancreatic growth hormone releasing factor (hpGRF) by recombinant DNA technique, using a nucleotide sequence encoding the entire hpGRF peptide sequence, a peptide segment linked to the amino terminal of the hpGRF peptide and a peptide segment linked to the carboxy terminal of the hpGRF peptide, said precursor being converted to GRF when appropriate processing enzymes are present in the transformed cells;
the European Patent Application Publication No. 0 129 073 which describes a process wherein a peptide with growth hormone releasing factor activity is prepared by cultivation of yeast transformed with DNA construct comprising a coding sequence encoding for growth hormone releasing factor and expressing a fused protein.